Interpretive Summary: Chronic alcohol consumption causes bad quality of bone and easier bone fracture, the mechanisms of how chronic alcohol effect on bone are largely unknown and potential treatments for prevention of alcohol-induced bone fracture remain unclear. In this report, we found that EtOH infusion for 4 weeks reduced bone quality of female rats. Chronic alcohol-induced inhibition of bone formation is associated with up-regulation of gene expression of nicotinamide adenine dinucleotide phosphate oxidase (Nox) in bone. These data suggest that inhibition of Nox expression or activity may be new target for prevention or treatment of chronic EtOH-induced bone loss, and perhaps other conditions resulting in oxidative stress associated bone resorption such as aging.

Technical Abstract:
Since the molecular mechanisms through which chronic excessive alcohol consumption induces osteopenia and osteoporosis are largely unknown, potential treatments for prevention of alcohol-induced bone loss remain unclear. We have previously demonstrated that, chronic ethanol (EtOH) treatment leads to accumulation of reactive oxygen species (ROS) in osteoblasts dependent on NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox). EtOH-induced ROS production might mediate both inhibition bone formation and increases in bone resorption. Using total enteral nutrition in a cycling female Sprague-Dawley rat model, we found that EtOH infusion for 4 weeks reduced bone mass (P less than 0.05) assessed by peripheral quantitative computerized tomography (pQCT) analysis. Co-administration of diphenylene iodonium chloride (DPI) a pan Nox inhibitor by daily s.c. injection of 1 mg/kg/d, abolished EtOH-induced bone loss. Static histomorphometric analysis revealed that EtOH effected both osteoblast and osteoclast indices (P less than 0.05). EtOH decreased bone volume and the number of osteoblasts, whereas it increased osteoclast number, bone surface covered by osteoclasts and the eroded bone surface characteristic of increased osteoclastic activity. DPI was able to normalize both osteoblast and osteoclast indices affected by EtOH. EtOH-induced bone loss was associated with up-regulation of mRNA levels of all three Nox subtypes 1, 2, 4 and RANKL (receptor activator of NF-kB ligand) in bone, and EtOH-induced RANKL promoter activity in vitro ST2 cell culture (P less than 0.05). To confirm Nox activation is truly associated with EtOH-induced bone loss, we fed a 36 percent alcohol containing liquid diet to nicotinamide dinucleotide phosphate oxidase-deficient p47phox-/- female mice for 6 weeks. In vivo systemic CT scan analysis revealed that, in wild type animals, EtOH reduced bone mineral density (BMD) compared to a pair fed group (P less than 0.05). In contrast, EtOH failed to down-regulate BMD in p47phox-/- female mice compared to their pair fed controls. Bone marrow cells taken from p47phox-/- female mice cultured with 50 mM EtOH for 24 h failed to induce RANKL gene expression but EtOH was able to induce RANKL gene expression in cells from wild type animals (P less than 0.05). These data suggest that inhibition of Nox expression or activity may be new target for prevention or treatment of chronic EtOH-induced bone loss, and perhaps other conditions resulting in oxidative stress associated bone resorption such as aging.